HIIE is known to induce transient inflammation and DNA damage in skeletal muscle (Jean et al., 2023). We have further found that the senolytic effect of exercise in human skeletal muscle occurs only at an intensity high enough to induce significant muscle inflammation (such as HIIE), as opposed to moderate intensity exercises with a similar amount of cycling work (Jean et al., 2023). This effect was evident 24 hours after exercise. Therefore, in this study we hypothesized that the senolytic effect of exercise is associated with transient inflammation during intense muscle contraction. To test this hypothesis, we measured the senolytic effect of HIIE under anti-inflammatory treatment to determine whether this effect may also occur as early as 3 hours after exercise. The key findings of the study are as follows: 1) A substantial decrease of cellular senescence in muscle occurred within 3 hours following an acute bout of HIIE and this senolytic effect persisted for 24 h; 2) HIIE notably decreased CD11b expression of immune cells in muscle tissues, suggesting that a decreased pro-inflammatory myeloid differentiation can occur within 3 hours following HIIE; 3) Ibuprofen administration delayed both the senolytic and anti-inflammatory effects of HIIE in human skeletal muscle. It is worthy to note that the observed senolytic and anti-inflammatory effects in human muscle were achieved by a very low volume of high intensity exercise (15 sets of 20-s cycling at 233 ± 11 watts) completed within 10 min and persisted for a long period (24 h) afterwards.
It is generally accepted that p16INK4a mRNA expresses only in replicable cells (He & Sharpless, 2017; Waaijer et al., 2012). In this study, the p16INK4a+ cells detected in IHC analysis may simply reflect the total number of mitotically active cells (such as stem cells). No p16INK4a+ signal was detected within myofibers supporting the notion that the nuclei within myofibers are post-mitotic (Yoshimoto, Ikemoto-Uezumi, Hitachi, Fukada, & Uezumi, 2020). Replenishment of damaged nuclei in myofibers are provided mostly from surrounding stem cells (Chikenji et al., 2019). Lowered p16INK4a mRNA in skeletal muscle without a change in p16INK4a+ cells suggests replenishment of new stem cells into exercised muscle tissues occurs within 3 hours after HIIE.
To further examine individual response among participants, we have found that the significant decrease in p16INK4a mRNA was contributed mainly by the muscle samples with high baseline senescence status. The muscle with low pre-exercise levels of senescence showed negligible changes in p16INK4a mRNA. This in vivo evidence suggests that exercise at high intensity provides a stringent selection pressure to those stem cells in the late stage of senescence. Furthermore, this outcome indicates that high intensity exercise can result in a dynamic shift of age profile of stem cells residing in human skeletal muscle. Whether this tissue renewal phenomenon is contributed by satellite cell division or bone-marrow stem cell replenishment remains to be examined (Dewi et al., 2023).
In this study, we have found a prolonged reduction in CD11b mRNA without significant declines in CD11b+ cells 3 hours following HIIE, suggesting a reduced proinflammatory myeloid differentiation of cells in skeletal muscle (Lagnado et al., 2021; Wang, 2018). Integrin CD11b is a surface marker presenting in many types of myeloid cells, particularly in neutrophils (Hynes, 1987). Myeloid cells can differentiate into both neutrophils and muscle stem cells depending on the tissue environment (Doyonnas, LaBarge, Sacco, Charlton, & Blau, 2004; Zhao et al., 2012). Neutrophils, featured by MPO expression, can specifically recognize and clear senescent cells by phagocytosis, forming an innate feedback mechanism to maintain youthfulness of tissues (Kay, 1975; Prata et al., 2018). The result of the present study, together with our previous findings (Jean et al., 2023), suggests that a longer period of anti-inflammatory response in skeletal muscle can be attained by a transient pro-inflammatory stimulus from a short episode of HIIE. Senescent cell accumulation is a known contributor to chronic inflammation, evidenced by a prominent increase in the secretion of proinflammatory mediators (Freund, Orjalo, Desprez, & Campisi, 2010). In this study, a very high correlation between p16INK4a mRNA and CD11b mRNA in skeletal muscle provides further support to this concept with this in vivo human evidence. The result of the study also confirms the previous finding (T. X. Y. Lee et al., 2021), in which a very large correlation exists between muscle cellular senescence marker p16INK4a mRNA and MPO mRNA in human skeletal muscle. MPO is highly expressed in phagocytes, suggesting lowered levels of inflammation following senescent cell clearance after high intensity exercise.
An acute bout of high intensity exercise can induce an immediate release of bone marrow stem cells into circulation (Agha et al., 2018). We have previously shown increased bone-marrow stem cell homing and proliferation in muscle tissues 24 hours after HIIE (Jean et al., 2023; T. X. Y. Lee et al., 2021). Chronic inflammation is currently known as a common denominator of age-associated metabolic disorders (Schafer, Miller, & LeBrasseur, 2017). Therefore, the prolonged anti-inflammatory response after a brief pro-inflammatory stimulus from HIIE suggests a clinical promise in treating metabolic complications. Despite ibuprofen’s ability to delay senolytic and anti-inflammatory effects following HIIE, the clinical concern is minimal as both placebo and ibuprofen achieved comparably low levels of cellular senescence and decreased CD11b expression after 24 hours of recovery.
DNA damage is a cause of cellular senescence, which can increase when the amount of DNA damage exceeds the body’s DNA repair capability (Chen, Hales, & Ozanne, 2007). We have recently shown concurrent increases in γ-H2AX+ myofibers and fragmented DNA levels in human skeletal muscle immediately after HIIE exercise (0 h) which was returned to the pre-exercise baseline within 24 hours (Jean et al., 2023). It was unknown whether the DNA integrity might be enhanced within a shorter period of time before 24 hours. γ-H2AX is a protein required for recruiting and localizing protein by forming DNA repair machinery (Kuo & Yang, 2008). In this study, we found no difference in γ-H2AX between pre-exercise and 3 hours post-exercise, suggesting that the DNA damage induced during HIIE may have recovered within 3 h. The results of the study are consistent with the previous observation that a complete recovery from acute exercise-induced DNA fragmentation can occur in 3 hours after moderate exercise (60-min exercise at 70% VO2max) (Wu et al., 2019). This fits well with the observed very low levels of in vivo cellular senescence within the same period in this study.
The present study provides the first evidence demonstrating the importance of acute inflammation on the senolytic effect of exercise. Inflammation is an immune program required for muscle regeneration after injury (Tidball, 2017). Ibuprofen is an anti-inflammatory drug widely used to reduce pain during and after exercise in training athletes. Whether a long-term use of ibuprofen has a negative impact on the positive senolytic effect from exercise remains to be examined. It has been shown that a high dose of ibuprofen (1200 mg/day similar to the present study) attenuated training adaptation in muscle strength and muscle hypertrophy following 8 weeks of resistance exercise in young adults (Lilja et al., 2018). However, a moderate-dose ibuprofen study showed negligible effect on muscle hypertrophy, muscle strength, and rating of muscle soreness following 6 weeks of resistance training in young adults (Krentz, Quest, Farthing, Quest, & Chilibeck, 2008). It is conceivable that rejuvenated tissues after senescent cell clearance have gained higher capability to extract nutrients compared with the tissues with greater proportion of senescent stem cells. It also remains to be seen whether the same inflammatory-mediating effects occur in the elderly consuming NSAIDs and exercising.